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. 2025 Feb 3;28(2):13. doi: 10.1007/s10456-024-09965-1

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Clioquinol binds to the ATP-binding pocket of VEGFR2 and causes its degradation. a Western blots showing VEGFR2 and β-actin expression in HUVECs treated with 0.1% DMSO (vehicle) or 10 µM clioquinol in the presence of 100 µM cycloheximide (CHX) for 0, 1, 2, 4, 6, and 8 h. b Expression level (% of 0 h) of VEGFR2 normalized to β-actin in HUVECs treated as described in (a) (n = 3 independent experiments). c mRNA level of VEGFR2 (% of control) in HUVECs treated with 0.1% DMSO (control) or 10 µM clioquinol for 4 h, as assessed by real-time PCR (n = 3). d Western blots showing VEGFR2 and β-actin expression in HUVECs that were pre-treated without or with 20 µM MG132 or 200 µM chloroquine (CQ) for 2 h and then exposed to 0.1% DMSO or 10 µM clioquinol for another 4 h. e Expression level (% of control) of VEGFR2 normalized to β-actin in HUVECs treated as described in (d) (n = 3 independent experiments). f Western blots showing VEGFR2 and β-actin expression in HUVECs that were pre-treated with 4 µg/mL IgG, 4 µg/mL anti-VEGFR2 NAb, 0.1% DMSO (vehicle), 100 nM lenvatinib, 250 nM tivozanib, or 1 mM ATP for 2 h and then exposed to 0.1% DMSO or 10 µM clioquinol for another 4 h. g, h Expression level (% of IgG or control) of VEGFR2 normalized to β-actin in HUVECs treated as described in (f) (n = 4 independent experiments). i Western blots showing p-VEGFR2, VEGFR2, and β-actin expression in HUVECs that were treated with 0.1% DMSO, 100 nM lenvatinib, 250 nM tivozanib or 10 µM clioquinol for 1 h and then stimulated with 25 ng/mL VEGF for 7 min. j Expression level (% of control) of p-VEGFR2 normalized to VEGFR2 in HUVECs treated as described in (i) (n = 4 independent experiments). k VEGFR2 kinase activity (% of control) in the presence of serial dilutions of clioquinol at an ATP concentration of 10 µM, as assessed by VEGFR2 kinase assay (n = 2). l VEGFR2 kinase activity (% of control) in the presence of lenvatinib (0.3, 1, and 3 nM) or clioquinol (10, 50, and 250 µM) at ATP concentrations of 10 or 500 µM, as assessed by VEGFR2 kinase assay (n = 3). Means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. (e, g, h, j: one-way ANOVA with Tukey’s multiple comparisons test; b, c, l: unpaired Student’s t-test)